Tuberculosis (TB) is one of the most important bacterial diseases of nonhuman primates (Adams et al., Biosafety in Nonhuman Primates in Biomedical Research: Biology and Management, (eds) Bennett, B. T., Abee, C. R. and R. Henrickson, Academis Press, San Diego, 1995). This is particularly true for captive nonhuman primate colonies, in which close quarters among colony members allow this highly contagious disease to spread rapidly. As in humans, most reported cases of this disease are caused by the acid-fast bacillus Mycobacterium tuberculosis or Mycobacterium bovis. And because these organisms are communicable between nonhuman primates and humans, nonhuman primate TB also poses a serious public health threat to humans that come into contact with infected animal colonies (Adams et al., 1995; Kalter et al., Develop. Boil. Standard 41:85-91, 1978).
Although the same TB organisms infect nonhuman primates and humans, morbidity and mortality rates are higher among nonhuman primates. This difference primarily stems from the distinct clinical manifestations of TB exhibited in humans and nonhuman primates. In humans, about 90% of all infected subjects are able to suppress active TB through cell-mediated immunity into a dormant- or latent-state. During the latent state of TB infection, the ability to spread the disease to others is very low and clinical symptoms are not apparent or nonexistent. Unlike humans, however, nonhuman primates do not exhibit a latent state. Once a nonhuman primate is infected, it presents with the active form of the disease, which it can readily spread to colony members and humans. Sadly, any infected nonhuman primates must either be isolated from its colony and humans or destroyed.
Because of the problems associated with nonhuman primate TB, careful surveillance for TB infections is very important for the welfare and good management of all captive nonhuman primates. In an effort to prevent TB outbreaks, all nonhuman primates are routinely tested for TB. At present, the standard means for detecting TB in nonhuman primates is tuberculin skin testing (TST). Unfortunately, TST has several shortcomings. Foremost, TST of nonhuman primates is extremely impractical. To test a nonhuman primate using TST, one must intradermally inject mammalian old tuberculin or tuberculin protein derivatives into the skin of an animal's upper eyelid. Because of the delicacy of this procedure, one must place an animal under deep chemical restraint or anesthesia during the injection. Following injection, one must check the eyelid at 24, 48, and 72 hours for a hypersensitivity-induced induration, the appearance of which indicates previous exposure to TB. Although one may also perform TST by intradermally injecting mammalian old tuberculin into the abdomen, abdominal injection is less desirable because it requires one to recapture the animal and palpate the test site. In contrast, induration at the eyelid causes the eyelid to swell and droop, a reaction that one may observe at a distance.
Another serious shortcoming of TST is that it lacks sensitivity. Sensitivity refers to the accuracy of a test, with which animals with a disease are correctly classified as positive. In nonhuman primates, the sensitivity of the TST is diminished by false negatives, or false negative results, reactions, or tests in which animals that do have TB fail to react as positive on the test. False negative tests are known to occur in early or advanced disease states. In advanced states, absence of delayed hypersensitivity has been attributed to anergy-absence of sensitivity to substances that would normally elicit an antigenic response. Concomitant severe illness, viral infections, such as measles, nutritional deficiencies, recent immunizations, or treatment with isoniazid—a crystalline antibacterial compound used to treat TB—may also result in false negative reactions. New world monkeys, including, but not limited to, squirrel monkeys, owl monkeys, and capuchins with TB have been reported to have a diminished hypersensitivity to tuberculin. Unfortunately, false negative tests are a major reason that TB outbreaks in nonhuman primates occur regularly in research colonies, zoos, and other facilities. These outbreaks disrupt expensive and important medical trials and lead to the destruction of many animals each year.
In addition to lacking sensitivity, TST also lacks specificity. Specificity refers to accuracy in correctly classifying a non-infected animal as negative or the absence of false positives, or false positive results, reactions, or tests in which animals that do not have TB react as positive on the test. Because of lack of specificity, TST results in numerous false positive skin tests each year. A major cause of false positive tests is believed to be cross-reactivity between some of the tuberculin antigens and nonpathogenic species of environmental mycobacteria. False positives are also known to arise from nonspecific inflammatory responses by an uninfected animal to a component of mammalian tuberculin (Butler et al., Medical Management: Biology and Management, (eds) Bennett, B. T., Abee, C. R., and R. Henrickson, Academis Press, San Diego, 1995; Corcoran and Jaax, Lab Anim. Sci, 41:57-62, 1991, Fox et al., J. Med. Primatol., 11:380-388, 1982). In some instances, false positives can even occur because of microtrauma to eyelid in the course of performing the intradermal injection.
False positive and false negative reactions have been a problem with all skin tests aimed at diagnosing TB in nonhuman primates. A review of the literature from 1970 to the present reveals dozens of papers citing instances of nonhuman primates that tested positive for TB by culture of M. tuberculosis or M. bovis organisms from body tissues—the gold standard of diagnosis—while repeatedly showing no delayed hypersensitivity to mammalian old tuberculin. Other mixtures of proteins, including Koch's Old Tuberculin, BCG, and various forms of PPD (purified protein derivatives) have been tried as skin test diagnostics in nonhuman primates. All had the same propensity for false positive and false negative results (Tauraso, Lab. Anim. Sci. 23:201-210, 1973; Corcoran and Jaax, 1991; Chaparas et al., Am. Ray. Resp. Dis. 112:43-47, 1975 Baram et al., Lab. Anim. Sci, 21:727-733, 1971; McLaughlin et al., Lab Anim. Sci, 26:44-50, 1976; Stunkard et al., Ant J. Vet. Res. 32:1873-1878, 1971).
Because of the propensity of TST to produce false positives and negatives, current testing protocols mandate multiple testing of each nonhuman primate imported into the United States. And given that approximately 9,000 nonhuman primates are imported each year, this is a daunting task. Upon entry into the United States, each of these 9,000 animals is usually held in quarantine for at least 90 days, in part because of the frequency and persistence of false negative TST reactions (Fox et al., 1982). While in quarantine, each animal is tested every two weeks and must have a minimum of five consecutive negative TB tests before being released from quarantine. If any animal tests positive, all other animals from the same shipment must begin a new period of quarantine (Butler et al., 1995). Animals that are released from quarantine, or captive-bred nonhuman primates that are maintained in colonies in which new animals are introduced, are generally tested for TB at least quarterly. Even closed colonies in which no new animals are introduced are tested at least once or twice per year in an effort to prevent TB outbreaks.
Recognizing the shortcomings of TST and the effort and costs associated with preventing TB outbreaks, several entities involved in the management of nonhuman primate colonies, including the Center for Disease Control, the NIH, and others, have called for the development of a quick and reliable test for nonhuman primate TB. In fact, for the past several years, the United States Public Health Service has solicited grant applications on the basis that an “urgent need currently exists for the development of improved methods for the detection of active tuberculosis in nonhuman primates.”
One recently developed test for nonhuman primate TB is PRIMAGAM™ (Pfizer Inc., New York, N.Y.). This test measures the cell-mediated immune response to a purified protein derivative (PPD), a crude antigen mixture that is similar to tuberculin. Specifically, two types of PPD—from M. Bovis and from M. avium—are used to trigger interferon-gamma production during TB infection. To measure interferon-gamma, PRIMAGAM™. relies on a multi-step enzyme immunoassay and spectrophotometry. Although PRIMAGAM™ is more specific than TST, it is less sensitive than TST (Garcia et al., 2004). For this reason, it has been recommended that PRIMAGAM™ be used in conjunction with TST (Garcia et al., Comp Med. 54(1):86-92, 2004).
Other tests have been developed to test for TB. For instance, U.S. Pat. No. 6,841,159 is directed to a lateral flow assay to detect M. tuberculosis and other mycobacteria. In the assay disclosed in U.S. Pat. No. 6,841,159, a nonspecific mixture of TB antigens is immobilized on a test strip. A sample of a patient's body fluid is then placed into contact with the non-specific mixture of antigens. If M. tuberculosis antibodies are present in the patient's sample, those antibodies form a visible antibody-antigen complex on the test strip, indicating a positive result. This test, however, is not specifically tailored to nonhuman primates. Thus, this test cannot account for animal-to-animal variations in antigen recognition patterns among nonhuman primate populations. Additionally, this test does not permit the detection of IgM antibodies in sufficient quantities to permit detection of TB antibodies early in nonhuman primate TB infection.
Accordingly, there is a need therefore for a TB test that is specifically designed for nonhuman primates. There is also a need for a nonhuman primate TB test that is less invasive than TST. Moreover, there is a need for a nonhuman primate TB test that is rapid and easy to administer. There is also a need for a nonhuman primate TB test that is more sensitive and more specific than TST. Additionally, there remains a need for a nonhuman primate TB test that needs only small amounts of test sample.